Relative neuronal vulnerability is a universal yet poorly understood feature of neurodegenerative diseases. In Parkinson's disease (PD), dopaminergic (DA) neurons in the substantia nigra (SN;A9) are particularly vulnerable, whereas adjacent DA neurons within the ventral tegmental area (VTA;A10) are essentially spared. We have previously demonstrated that molecular differences between these DA neurons contribute to their differential vulnerability 1 and can be utilized to protect against neurodegeneration in vivo 2. Here we propose to study the monomeric GTPase protein RAB3B that is highly enriched in VTA (A10) compared to SN (A9) both in rat and human midbrain. RAB3B is localized to synaptic vesicles in neurons and is known to regulate neurotransmitter exocytosis by regulating synaptic vesicle trafficking processes including docking, fusion, and recycling at the synaptic terminals. RAB3B also activates phosphoinositide-3-kinase (PI3K) that phosphorylates and activates the pro-survival kinase Akt. These functions are of particular interest because, first, disruption of vesicle trafficking and synaptic dysfunction have been strongly implicated in toxicity induced by a-synuclein, a protein that aggregates in PD and that has been genetically linked to the disease;second, Akt signaling has previously been shown to protect against oxidative stress, a process thought to play an important role in PD pathogenesis and that is exploited in a variety of toxic PD models. Our preliminary data strengthen the rationale for our grant proposal by demonstrating that: 1) RAB3B increases the levels of synaptic proteins in addition to key regulators of dopamine homeostasis in vitro;2) RAB3B increases dopamine content in vitro and in vivo;3) RAB3B protects against toxins that simulate aspects of PD in vitro, including the oxidative stressor 6-hydroxydopamine (6-OHDA) and the proteasome inhibitor MG-132;4) a prominent early change preceding DA neuron loss in a rat adeno-associated virus-2 (AAV2) synucleinopathy model includes alteration of synaptic proteins. On the basis of the native biological function of RAB3B and our preliminary data, we hypothesize that RAB3B contributes to the reduced vulnerability of VTA (A10) DA neurons in PD and that overexpressing RAB3B will protect vulnerable SN (A9) DA neurons in vivo. By utilizing the AAV2 system, we propose to delineate the biological effects of RAB3B expression in the rat SN in vivo, with a particular emphasis on processes critical for synaptic function including vesicular transport and neurotransmitter release and storage. We will then test whether RAB3 expression in vivo protects SN (A9) neurons in two complementary rodent models of PD: AAV2-mediated 1-synuclein overexpression and a 6-OHDA induced oxidative stress lesion. References 1. Chung, C.Y., et al. Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. Hum Mol Genet 14, 1709-1725 (2005). 2. Chung, C.Y., Koprich, J.B., Endo, S. &Isacson, O. An endogenous serine/threonine protein phosphatase inhibitor, G-substrate, reduces vulnerability in models of Parkinson's disease. J. Neurosci 27, 8314-8323 (2007). PUBLIC HEALTH RELEVANCE In Parkinson's disease (PD), dopaminergic (DA) neurons in the substantia nigra (A9) are considerably more susceptible than the immediately adjacent DA neurons in the ventral tegmental area (A10) and this differences between these neuronal populations may be useful in designing novel therapeutic and neuroprotective strategies for PD. RAB3B is a molecule more highly expressed in A10 DA neurons and its function in regulating synaptic vesicle trafficking, a process disrupted in PD, makes it an exciting candidate neuroprotective agent. Our goal in this grant is to characterize the neuroprotective role of RAB3B in two different rodent models of Parkinson's disease.